Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Protocolo

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Immunohistochemistry

1. Thymidine analog staining​
   1. Place tissue sections in PBS (phosphate buffered saline) and mount 5−8 sections on a positively charged slide, maintaining serial order from anterior to posterior and the same orientation. Let tissue sections dry to completely adhere to slides, and then draw a border using a hydrophobic pen or PAP pen.
   2. Permeabilize sections with permeabilization buffer (0.5% TBS (tris buffered saline) -triton) for 20−30 min. Then wash sections 2x using TBS-triton for 5 min each.
      ​NOTE: Permeabilization aids intracellular antibody penetration. Permeabilization time can be adjusted depending on tissue thickness and antibody efficiency. Alternatively, one can increase detergent concentration to increase permeabilization potency. However, care should be taken to not permeabilize for too long since tissue fragility increases with prolonged permeabilization time.
   3. Prepare Edu (5-ethynyl-2'-deoxyuridine) reaction solution according to Table 1. The final concentration of Alexa488-azide is 15 µM in 1 mL of Edu reaction solution.
   4. Incubate sections in the Edu reaction solution for 30 min to 1 h and then wash 3x in TBS-triton for 5 min each. After this step, cover slides in aluminum foil to protect them from light. At this stage, check if the Edu reaction works by using a fluorescent microscope. Edu-labeled cells will fluoresce under an epifluorescence microscope.
2. Mounted tissue section protocol
   1. Block tissue sections mounted on a slide from step 1.1.4 using blocking buffer raised in the same animals as the secondary antibody, e.g., donkey serum, for 30 min to 1 h and then wash 2x in TBS-triton for 5 min each.
   2. Prepare primary antibody (i.e., chicken anti-nestin) solution during the blocking step by mixing primary antibodies in blocking buffer solution at 1:200. 250 µL per slide is sufficient to ensure that tissue is completely submerged in solution.
   3. Incubate tissue sections in a primary antibody solution overnight at RT after blocking washes. Modify this step depending on the primary antibody used.
      NOTE: If antibodies have a high background or non-specific binding, incubating at 4 °C instead of RT (room temperature) may improve results. Antibodies with poor tissue penetration can be left to incubate for 2 days if needed.
   4. Incubate tissue sections 3x in TBS-triton for 5 min to remove excess primary antibody. Then, incubate tissue sections in fluorophore-conjugated secondary antibodies (e.g., Alexa 647 anti-chicken at 1:200) prepared in blocking buffer solution for 2 h at RT.
   5. Incubate tissue sections 3x in TBS-triton for 5 min to remove excess secondary antibody and apply 300 µM DAPI (4′,6-diamidino-2-phenylindole) solution at 1:100 in PBS for 15 min at RT.
   6. Incubate tissue sections 3x in PBS for 5 min to remove excess DAPI and remove the pap pen circle from around the tissue using a cotton swab or a delicate task wipe. Let sections dry, and then apply mounting media and coverslip. Let mounting media dry before imaging slides.

Table 1: Solutions utilized for immunohistochemistry

Antifreeze Solution	Ethylene-glycol 150 mL + sucrose 150 g + fill to 500 mL 0.1 M PB for 500 mL solution
Citrate Buffer	9 mL of citric acid stock + 41 mL of tri-sodium citrate buffer + 450 mL of ddH2O
Citric acid stock	[0.1 M] Citric Acid 21 g/1 L ddH2O
Tri-sodium citrate stock	[0.1 M] Tri-sodium Citrate 29.4 g/1 L ddH2O
Tris Buffered Saline -Triton (TBS -Triton)	0.05% 100-x Triton in TBS
Permeabilization Buffer	0.5% 100-x Triton in TBS
Blocking Buffer	0.33 mL Donkey Serum in 10 mL TBS-Triton
Edu Reaction Solution	Make a CuSO4·5H2O solution by adding 1 mg of CuSO4·5H2O in 4 mL solution of [0.1 M] Tris pH 8.5. Then add 1:40 of a 600 µM Alexa488-azide solution and 10 mg/mL of L-Na+ ascorbate to the CuSO4·5H2O solution before applying it to tissue.

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Materiais

Name	Company	Catalog Number	Comments
5-Ethynyl-2'-deoxyuridine (Edu)	Carbosynth	NE08701
Alexa-488 Azide	Thermo Fisher Scientific	A10266
Anti-Chicken Nestin	Aves	NES; RRID: AB_2314882
Anti-Goat DCX	Santa Cruz	Cat# SC_8066; RRID: AB_2088494
Anti-Mouse Tbr2	Thermo Fisher Scientific	14-4875-82; RRID: AB_11042577
Confocal Software (Zen Black)	Zeiss Microscopy	Zen 2.3 SP1 FP1 (black)
Copper (II) Sulfate Pentahydrate	Thermo Fisher Scientific	AC197722500
Coverslip	Denville Scientific	M1100-02
Plus Charged Slide	Denville Scientific	M1021
Phosphate Buffered Solution (PBS)	Thermo Fisher Scientific	10010031
Super PAP Pen 4 mm tip	PolySciences	24230
Tris Buffered Solution (TBS)	Sigma-Aldrich	T5912
Triton X-100	Sigma-Aldrich	93443